Extracellular recording electrode

ABSTRACT

A multiple electrode includes a plurality of micro-electrodes provided on a substrate, and a wiring portion for providing an electrical signal to the micro-electrodes or extracting an electrical signal from the micro-electrodes. Each micro-electrode has porous conductive material on its surface, and the impedance of the micro-electrode is 50 kΩ or less. Preferably, the porous conductive material is gold, and formed by the passage of current at a current density of 1.0 to 5.0 A/dm 2  for 10 to 360 sec. The multiple electrode may include micro-electrodes provided on a substrate in the form of a matrix, a lead line connected to the micro-electrodes, and an electrical junction connected to an end of the lead line.

TECHNICAL FIELD

[0001] The present invention relates to a multiple electrode forextracellular recording, which is useful in the field ofelectro-physiology and is used to measure changes in potentialassociated with the activity of neurons.

BACKGROUND ART

[0002] Recently, the applicability of neurons to electronic devices hasbeen vigorously studied as well as the medical study. An actionpotential is generated in a neuron which is in an active state. A changein the ion permeability of a neuron leads to changes in intra- andextracellular ion concentrations which are responsible for generation ofan action potential. Therefore, if a potential change in associationwith a change in ion concentration around a neuron is measured, theactivity of the neuron can be monitored.

[0003] The above-described potential measurement utilizing cell activityis conventionally conducted by placing an electrode of glass or metal(e.g., platinum) for measuring an extracellular potential around a cellwith the aid of a micro-manipulator or the like. Alternatively, asimilar electrode is inserted into a cell so as to measure theelectrical activity of the cell. These conventional techniques have thefollowing disadvantages: skill in electrode preparation is required; theelectrode has high impedance and therefore the signal is susceptible toexternal noise; and cells or tissues are injured if an electrode isinserted into the cell. Therefore, conventional electrodes are notsuitable for long-term monitoring.

[0004] To avoid such problems, the inventors have developed a multipleelectrode including a plurality of micro-electrodes made of a conductivematerial provided on an insulating substrate, and a lead pattern, onwhich cells or tissue can be cultured (Japanese Laid-Open PublicationNo. 6-78889, and Japanese Laid-Open Publication No. 6-296595). With thismultiple electrode, the activities of neurons can be monitored withoutinjuring cells or tissue for a long period of time.

[0005] In the above-described multiple electrode, an uppermost surfaceof the electrode contacting cells is plated with porous platinum blackusing electrolysis (Japanese Laid-Open Publication No. 6-78889), or withgold using deposition (Japanese Laid-Open Publication No. 6-296595). Inthe case of the platinum black plating, although it is easy to adjustthe impedance of the electrode to a practical level, i.e., about 50 kΩor less, the strength of the electrode is low and therefore therecyclability of the electrode is low. In the case of gold formed bydeposition, the strength is improved, but it is difficult to reduce theimpedance to about 50 kΩ or less.

DISCLOSURE OF THE INVENTION

[0006] The present invention is intended to solve the above-describedproblems. The object of the present invention is to provide anextracellular recording electrode having impedance-frequencycharacteristics suitable for recording an electrical signal of a cell,which has a low level of impedance, insusceptability to external noise,a high strength, and ease in recycling electrodes.

[0007] The inventors have found that in production of an extracellularrecording electrode, the current density used in forming a conductivematerial on an uppermost surface thereof is optimized, thereby obtaininga porous conductive material surface which is rough and therefore has anincreased surface area, and that the porous conductive material haspreferable characteristics for an extracellular recording electrode. Thepresent invention has been completed based on the above-describedfindings.

[0008] The present invention provides a multiple electrode for measuringelectro-physiological characteristics of a cell. The electrode includesa plurality of micro-electrodes provided on a substrate, and a wiringportion for providing an electrical signal to the micro-electrodes orextracting an electrical signal from the micro-electrodes. Themicro-electrodes have a porous conductive material on a surface thereof,the conductive material is selected from the group consisting of gold,titanium nitride, silver oxide, and tungsten, and the impedance of eachof the micro-electrode is 50 kΩ or less.

[0009] Preferably, the porous conductive material is gold, and isprovided by passage of current at a current density of 1.0 to 5.0 A/dm²for 10 to 360 sec.

[0010] The present invention also provides a multiple electrode formeasuring electro-physiological characteristics of a cell. The electrodeincludes a plurality of micro-electrodes provided on a substrate, and awiring portion for providing an electrical signal to themicro-electrodes or extracting an electrical signal from themicro-electrodes. The surface area of the micro-electrode calculatedfrom an electrostatic capacity of an equivalent circuit havingsubstantially the same impedance as that of the micro-electrode, isgreater than or equal to at least 10 times and less than 200 times theprojection area of the micro-electrode, and the impedance of each of themicro-electrode is 50 kΩ or less.

[0011] The term “projection area of a micro-electrode” as used hereinrefers to the entire area of an uppermost surface of a micro-electrodebefore a conductive material is provided.

[0012] Preferably, a surface area of the micro-electrode measured by agas adsorption method is less than or equal to 5×10⁵ times theprojection area of the micro-electrode.

[0013] In one embodiment of this invention, the micro-electrodes arearranged on the substrate in a form of a matrix, the wiring portionincludes a lead line connected to the micro-electrode and an electricaljunction connected to an end of the lead line, and at least a surface ofthe lead line is covered with an insulating layer.

[0014] In one embodiment of this invention, the porous conductivematerial may be provided by etching, such as RIE (reactive ion etching)and ICPRIE (inductively coupled plasma RIE).

[0015] The present invention also provides an integrated cell installerincluding the above-described multiple electrode. The integrated cellinstaller has a cell installing region for placing a cell or tissue onthe substrate of the multiple electrode.

[0016] The present invention also provides a cellular potentialmeasuring apparatus including the above-described integrated cellinstaller, an output signal processor connected to the micro-electrodesfor processing an output signal due to an electro-physiological activityof a cell or tissue, and a stimulus signal provider for optionallyproviding an electrical stimulus to the cell or tissue.

[0017] The present invention also provides a cellular potentialmeasuring system including the above-described cellular potentialmeasuring apparatus, an optical monitoring apparatus for opticallymonitoring a cell or tissue, and/or a cell culture apparatus forcontrolling the culture environment of the cell or tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1a is a photomicrograph showing a gold plating formed byelectrolysis on a surface of a micro-electrode at a current density of1.0 A/dm² in a comparative example where a magnification is 2500. Thescale bar in the figure is 50 μm.

[0019]FIG. 1b is a photomicrograph showing a gold plating formed byelectrolysis on a surface of a micro-electrode at a current density of1.5 A/dm² according to the present invention. The scale bar in thefigure is 50 μm.

[0020]FIG. 1c is a photomicrograph showing a gold plating formed byelectrolysis on a surface of a micro-electrode at a current density of2.0 A/dm² according to the present invention. The scale bar in thefigure is 50 μm.

[0021]FIG. 2a is a diagram showing a printout of a computer screendisplaying 64 channels of potential change responses of cells ongold-plated micro-electrodes obtained by electrolysis at a currentdensity of 1.0 A/dm² in the comparative example with respect to aconstant current stimulus. A stimulus signal is applied to a channel 29.

[0022]FIG. 2b is a diagram showing a printout of a computer screendisplaying 64 channels of noise levels of the gold-platedmicro-electrodes shown in FIG. 2a in the absence of cells.

[0023]FIG. 2c is a diagram showing a printout of a computer screendisplaying 64 channels of potential change responses of cells ongold-plated micro-electrodes obtained by electrolysis at a currentdensity of 1.5 A/dm² according to the present invention with respect toa constant current stimulus.

[0024]FIG. 2d is a diagram showing a printout of a computer screendisplaying 64 channels of noise levels of the gold-platedmicro-electrodes shown in FIG. 2c in the absence of cells.

[0025]FIG. 2e is a diagram showing a printout of a computer screendisplaying 64 channels of potential change responses of cells ongold-plated micro-electrodes obtained by electrolysis at a currentdensity of 2.0 A/dm² according to the present invention with respect toa constant current stimulus.

[0026]FIG. 2f is a diagram showing a printout of a computer screendisplaying 64 channels of noise levels of the gold-platedmicro-electrodes shown in FIG. 2e in the absence of cells.

[0027]FIG. 3 is a diagram showing an impedance characteristic of themicro-electrode of the present invention.

[0028]FIG. 4a is a diagram showing an equivalent circuit of themicro-electrode of the present invention.

[0029]FIG. 4b is a diagram showing an equivalent circuit of themicro-electrode of the present invention.

[0030]FIG. 5 is a diagram showing an impedance characteristic of anequivalent circuit of the micro-electrode of the present invention.

[0031]FIG. 6 is a diagram showing an impedance characteristic of anequivalent circuit of the micro-electrode of the present invention.

[0032]FIG. 7 is a diagram showing an impedance characteristic of amicro-electrode of a comparative example.

[0033]FIG. 8 is a diagram showing an impedance characteristic of anequivalent circuit of a micro-electrode of a comparative example.

[0034]FIG. 9 is a diagram showing an impedance characteristic of amicro-electrode of a comparative example.

[0035]FIG. 10 is a diagram showing an impedance characteristic of anequivalent circuit of a micro-electrode of a comparative example.

[0036]FIG. 11 is a graph showing a result of a lifetime test in which anelectrolytic gold-plated micro-electrode of the present invention iscompared with a conventional product in terms of reusability.

BEST MODE FOR CARRYING OUT THE INVENTION

[0037] Hereinafter, the present invention will be described in moredetail.

[0038] (Production of Porous Conductive Material for Micro-electrode)

[0039] A multiple electrode for extracellular recording according to thepresent invention includes a plurality of micro-electrodes provided onan insulating substrate. Cells are placed on the micro-electrodes tomeasure the electrical activity of the cells.

[0040] The multiple electrode of the present invention particularlyincludes a porous conductive material on an uppermost surface of themicro-electrode. The impedance of this micro-electrode is 50 kΩ or less.The impedance of the micro-electrode is preferably 35 kΩ or less, morepreferably 25 kΩ or less, and even more preferably 10 kΩ or less. Animpedance as used herein is defined as a value which is measured at afrequency of 1 kHz and at an interterminal voltage of 50 mV. The lowerlimit value of the impedance in the present invention is notparticularly limited, but is preferably as low as possible in accordancewith the teachings of the present invention.

[0041] It is believed that such a low impedance is attributed to aporous structure of a conductive material provided on the electrode. Theporosity as used herein refers to the state of a conductive materialsurface being rough or having minute protrusions and depressions. Whenmagnified and observed by an optical microscope, the porous conductivematerial surface of the present invention appears to be a denseagglutination of about 0.01-25 μm-diameter small particles. When theuppermost surface of the electrode has such a porous structure, thesurface area is significantly increased. As a result, a low level ofimpedance can be achieved which cannot be otherwise obtained in the caseof a smooth gold surface which is provided by conventional deposition.

[0042] As described above, the porous structure of the uppermost surfaceof the electrode can be defined by the surface area of the uppermostsurface of the electrode. The surface area of the uppermost surface ofthe electrode may be defined with a BET method using gas absorptionwhich is well known to those skilled in the art, for example.Alternatively, the impedance may be calculated based on theelectrostatic capacity of an equivalent circuit of an electric circuitrepresenting a model of an interface between a micro-electrode and asolution.

[0043] The porous conductive material of the present invention isrepresentatively produced by electrolytic plating under over-currentdensity. The over-current density preferably refers to a current densityin the range of 1.2 A/dm² ₁ more preferably 1.0 to 5.0 A/dm², and mostpreferably 1.4 to 2.1 A/dm². This is contrast to typical industrialelectrolytic plating for a conductive material where the current densityis about 1.0 A/dm² or less. Note that even when a current density ofmore than 3.0 A/dm² is used, the porous conductive material plating ispossible. If a current density is excessively large, the surface isextremely rough so that it is difficult to maintain an intended shape(e.g., square) of a micro-electrode. In the present invention,conductive material plating can be produced by the passage of currentfor representatively 10-360 seconds, preferably 30-240 seconds, underover-current density. If the passage time of the current is excessivelyshort, a conductive material plating may not be sufficiently formed on amicro-electrode. If the passage time of the current is excessively long,the growth of a conductive material on a micro-electrode is not even, sothat some portions of the conductive material plating is rapidly grownwhile others are slowly grown and the shape of the electrode is likelyto deviate from a square.

[0044] The above-described conditions for electrolytic plating are onlyfor illustrative purposes, and may be optionally modified within a rangein which the above-described low impedance can be achieved, depending onthe limitations of the electrolytic plating apparatus used orrequirements of operational procedures.

[0045] Alternatively, the porous conductive material of the presentinvention can be obtained by etching. For example, chemical etchingusing an oxidizing agent and a solubilizing agent, electrochemicaletching in which electrolysis is conducted using direct or alternatingcurrent in an electrolyte solution containing acid as a major component,or the like can be employed, whereby the surface area can be increased.

[0046] The uppermost surface of the micro-electrode of the presentinvention has the above-described porous structure. Further, a materialfor the uppermost surface is, for example, gold, whereby a high strengthas well as low impedance characteristics can be achieved. Therefore, therecycling efficiency of the micro-electrode is high, resulting in highcost performance. This is contrast to an electrolytic plating ofplatinum black heavily used in conventional electrodes which has a lowlevel of impedance, but a significantly low level of strength, so thatit is not resistant to repetitions of recycling. Specifically, the rateof increase in the impedance of the electrode of the present inventionwith respect to the original impedance after 20 cycles isrepresentatively 30% or less, preferably 20% or less, and morepreferably 15% or less, when a lifetime test is conducted undersubstantially the same conditions as those in Example 5 described later.

[0047] The above-described porous conductive material is provided on theuppermost surface of the micro-electrode of the present invention. Anunderlying electrode material for the porous conductive material may beany material which can be sufficiently adhered to the porous conductivematerial. Examples of an underlying electrode material for the porousconductive material include, but are not limited to, preferably nickelby electroless or electrolytic plating, gold by commonly usedelectroless plating, and the like. The thickness of these underlyinglayers is not particularly limited. For example, the thickness of thenickel plating is about 3000 to 7000 angstroms. Electroless gold platinghaving a thickness of about 300 to 700 angstroms may be provided on thenickel plating.

[0048] In the multiple electrode of the present invention,representatively, a plurality of micro-electrodes are provided on asubstrate in such a manner as to be placed at intersections of a gratingin the form of a matrix. In this arrangement, a plurality of electrodescan be equally spaced. Therefore, the cell bodies of adjacent neuronscan be placed on adjacent electrodes to detect transfer of an electricalsignal between the cell bodies.

[0049] Each micro-electrode is externally provided with an electricalsignal. Alternatively, a wiring portion for extracting an electricalsignal from each micro-electrode to the outside is connected to eachelectrode. Representatively, the wiring portion includes a lead linewhich is connected to each micro-electrode and drawn from the electrodetowards the periphery of the substrate. The wiring portion may furtherinclude an electrical junction connected to an end of the lead linewhich is typically located at the periphery of the substrate. An exampleof a material for the wiring portion preferably includes indium tinoxide (ITO). Note that the above-described impedance is an overallcharacteristic value of the micro-electrode and the wiring portion. Infact, the impedance value of the above-described wiring portion isnegligible compared to the value defined by the material and dimensionsof the uppermost surface of the electrode. Therefore, selection ofmaterials for the underlying layer of the electrode and the wiringportion substantially does not have an influence on the impedance.

[0050] Representatively, the surface of a lead line is covered with aninsulating layer. The insulating layer may be provided only on the leadline, but preferably on almost the entire upper surface of the substrateexcept for the micro-electrodes and the vicinity of the electricaljunctions. Examples of the insulating layer preferably include acrylicresin or photosensitive polyimide which are easy to process.

[0051] (Configuration of Multiple electrode)

[0052] For the detailed design of the multiple electrode of the presentinvention, any structural features of a known multiple electrode (e.g.,Japanese Laid-Open Publication No. 6-78889) can be used as long as theformation and function of the above-described porous conductive materialare not interfered with. Hereinafter, a configuration of arepresentative example of the multiple electrode will be shown.Embodiments as described herein may be optionally modified by takinginto consideration various factors, such as the characteristics ofneurons to be measured, the nature of data to be measured, and the like.

[0053] The substrate included in the multiple electrode is preferablymade of a transparent insulating material for the purpose of opticalmonitoring after cell culture. Examples of such a material include:glass, such as silica glass, lead glass, and borax glass; an inorganicsubstance, such as quartz; polymethylmethacrylate or a copolymer; and atransparent organic substance, such as polystyrene, and polyethyleneterephthalate. An inorganic substance which has mechanical strength andtransparency is preferable.

[0054] Examples of a material for the electrodes provided on thesubstrate include indium tin oxide (ITO), tin oxide, Cr, Au, Cu, Ni, Al,and Pt. Among other things, ITO and tin oxide are preferable. ITO havingtransparency and a high level of conductivity is particularlypreferable. The above-described micro-electrode is typically produced byproviding the porous conductive material plating on the uppermostsurface of a part of the electrode material having a desired positionand shape.

[0055] Typically, a plurality of micro-electrodes are equally spaced insuch a manner that the distances between adjacent electrodes are allequal to each other. The distances between adjacent electrodes may berepresentatively in the range of from about 10 to about 1000 μm.Representatively, the shape of the electrode is substantially a squareor a circle where an edge or a diameter is within the range of fromabout 20 to about 200 μm. With the above-described settings, if the cellbody of a neuron (i.e., a cell body, a dendrite, and an axon) to bemeasured is located on an electrode, it is highly probable that anothercell body, to which a dendrite of the former cell body is connected, islocated on an adjacent electrode.

[0056] A lead line connected to the micro-electrode may be made of thesame electrode material as those described. In this case, ITO is alsopreferable. Typically, such an electrode material is deposited on asubstrate. Thereafter, etching is conducted using a photoresist, therebyforming a desired integrated pattern of a lowermost layer of themicro-electrode and a wiring portion including a lead line. In thiscase, the thicknesses of the lowermost layer of the micro-electrode andthe wiring portion may be about 500 to 5000 angstroms.

[0057] The lead line is representatively arranged extendingsubstantially radially from each micro-electrode. In combination withthis substantially radial arrangement, a plurality of micro-electrodesare particularly preferably arranged in such a manner that the centersthereof are placed on respective intersections of an 8×8 grating.

[0058] An example of a material for an insulating layer covering thelead line includes a transparent resin, such as a polyimide (PI) resinand an epoxy resin. A photosensitive resin, such as negativephotosensitive polyimide (NPI), is preferable. For example, when aphotosensitive resin is used as the insulating layer material, it ispossible to expose only the electrode by forming an opening in theinsulating layer portion on the micro-electrode by utilizing a patternformed by photoetching. As described above, the insulating, layer isprovided in such a manner as to cover substantially the entire surfaceof the insulating substrate except for the vicinity of the electrodesand the electrical junctions with external circuits. This is preferablein terms of production efficiency and the like.

[0059] (Apparatus and System for Measuring Cellular Potential)

[0060] For the detailed design of various components of a system foreffectively utilizing the multiple electrode of the present inventionfor measuring neurons or the like, any structural features of a knownsystem for measuring a cellular potential (e.g., Japanese Laid-OpenPublication No. 8-62209) can be adopted as long as the formation andfunction of the above-described porous conductive material are notinterfered with.

[0061] Typically, the multiple electrode of the present invention isadditionally provided with a structure for facilitating cell culture tobe conducted on the multiple electrode and optionally with anotherstructure for facilitating handling of the multiple electrode. Theresultant multiple electrode may be provided as an integrated cellinstaller.

[0062] In order to conduct cell culture on the multiple electrode,representatively, a structural member capable of holding culture mediummay be provided via the insulating layer on the substrate which issubstantially entirely covered with the insulating layer. For example, acylinder-like frame made of polystyrene may be fixed on the substrate insuch a manner as to surround a plurality of micro-electrodes, therebyobtaining the above-described holding structure. In this case, theinside of the polystyrene frame defines a cell holding region. Theporous conductive material plating of the present invention may beformed on the surface of the micro-electrode before or after theprovision of the holding structure.

[0063] In order to facilitate the handling of the multiple electrode inmeasuring cells, for example, a printed circuit board may be used. Theprinted circuit board has a conductor pattern conductively connected toelectrical junctions on the multiple electrode, thereby playing a rolein extending an electrical connection, which is established from themicro-electrode to the electrical junction, to the outside. A holderhaving an appropriate shape, such as a two-part split holder whichsandwiches the multiple electrode, may be used to reliably fix theprinted circuit board with the multiple electrode while keeping theelectrical connection therebetween, for example.

[0064] The integrated cell installer may be further combined with astimulus signal provider and an output signal processor, therebyobtaining a cellular potential measuring apparatus for electricallystimulating cells on the multiple electrode, and processing an outputsignal which is a response to the stimulus.

[0065] The stimulus signal provider can apply a stimulus signal to anypair of electrodes out of the plurality of micro-electrodes. When a cellresponds to the stimulus signal, another electrode detects a change inevoked potential and outputs an output signal corresponding to thechange to a signal processor. The output signal is transferred via anappropriate process to a display apparatus or the like, for example.Note that a spontaneous potential generated in a cell without receivinga stimulus signal may be similarly measured.

[0066] The stimulus signal provider and the output signal processor arerepresentatively realized by a single computer having appropriatemeasurement software. The measurement software provides, on a computerscreen, a parameter setting window for setting stimulus conditions andthe like, a recording window for recording a potential change detectedfrom a cell and displaying the data via multiple channels in real time,a data analyzing window for analyzing recorded data, and the like.Preferably, a stimulus signal from a computer is transferred via a D/Aconverter to the multiple electrode, while an output signal from a cellis transferred via an A/D converter to a computer.

[0067] A cellular potential measuring apparatus may be further combinedwith an optical monitoring apparatus and a cell culture apparatus,thereby obtaining a cellular potential measuring system for culturingneurons for a long period of time, and stably and accurately measuringthe electro-physiological activities of the neurons. The opticalmonitoring apparatus may include an inverted microscope, and further anSIT camera for a microscope including a high-definition display and animage file apparatus. As the cell culture apparatus, any apparatus orcombination thereof which can control the temperature of the cultureatmosphere, the circulation of culture medium, the supply of a gasmixture of air and carbon dioxide, and the like, can be used.

EXAMPLES

[0068] Hereinafter, the present invention will be specifically describedby way of illustrative examples. The present invention is not limited tothese examples.

Example 1

[0069] A surface of a planar multiple electrode was coated withelectrolytic gold plating under various current densities (a centralportion of each electrode having a size of 50×50 μm was positioned atone of intersections of an 8×8 grating. Therefore, the entire surfacearea of the micro-electrode (projection area) was 50×50×64=160000 μm²).

[0070] Specifically, electrolytic gold plating was formed at a currentdensity of 1.0 A/dm², 1.5 A/dm², and 2.0 A/dm². The impedance of themicro-electrode having the gold plating formed at the respective currentdensities was measured under conditions of a frequency of 1 kHz, aninterterminal voltage of 50 mV, and the average of five measurements wascalculated. The results are shown in Table 1. As the current density wasincreased, the average impedance of each of the micro-electrode could belowered. TABLE 1 Current Density and Average Impedance Current density(A/dm²) Average impedance (kΩ) 1.0 336.39 ± 78.59 1.5  22.07 ± 1.95 2.0 16.56 ± 2.34

[0071] The gold-plated surface of the micro-electrode was observed withan optical microscope. The photomicrographs are shown in FIGS. 1a to 1c. FIGS. 1a, 1 b, and 1 c are microphotographs of the plated surfaceswhich were obtained at a current density of 1.0 A/dm², 1.5 A/dm², and2.0 A/dm², respectively. In the case of 1.0 A/dm² current density, asubstantially smooth plated surface was obtained. In contrast, as thecurrent density was increased, the porosity of the gold-plated surfacewas more significant and the area of the electrode surface wasincreased.

Example 2

[0072] The micro-electrode surfaces obtained in Example 1 and slices ofthe mouse hippocampus (brain) were actually used to measure evokedpotentials and noise levels. Hippocampus slices were obtained from amouse. A five-week-old male c57black6 mouse was anesthetized withFluothane and decapitated to remove a whole brain. The removed brain wasimmediately cooled in Ringer's solution on ice. A brain block containingonly the hippocampus was dissected. Thereafter, the obtained brain blockwas cut by a tissue slicer to give a slice having a thickness of 250 μm.The slice was placed and tested on the micro-electrodes.

[0073] Evoked potentials and noise levels were measured in the presenceof an applied constant current of 10 μA having bipolar pulses (where thepulse width is 100 μsec). Responses of 64 electrodes were measured from5 msec before stimulation to 45 msec after the stimulation, and weredisplayed on a computer screen having 64 channels. The results are shownin FIGS. 2a to 2 f. FIG. 2a, 2 c and 2 e show potential change responses(i.e., evoked potentials) of cells on the gold-plated micro-electrodewith respect to the above-described constant current stimuli. FIG. 2ashows evoked potentials of the electrode obtained by electrolysis at acurrent density of 1.0 A/dm². FIG. 2c shows evoked potentials of theelectrode obtained by electrolysis at a current density of 1.5 A/dm².FIG. 2e shows evoked potentials of the electrode obtained byelectrolysis at a current density of 2.0 A/dm².

[0074]FIGS. 2b, 2 d and 2 f show noise levels occurred in the respectivegold-plated micro-electrodes shown in FIGS. 2a, 2 c and 2 e.

[0075] As can be seen from FIGS. 2a, 2 c and 2 e, the electrodes havingthe porous gold plating obtained by a higher level of current densityhad clear responses to the applied stimulus signals and it is possibleto apply a constant current stimulus to the electrodes in an effectivemanner, as compared to the gold plating (FIG. 2a) obtained byelectrolysis at a current density of 1.0 A/dm² (FIGS. 2c and 2 e).Particularly, the micro-electrode having plating obtained at a currentdensity of 1.5 A/dm² had a low impedance value and a satisfactorysurface state.

[0076] As to noise level, the plating (FIG. 2f) obtained at a currentdensity of 2.0 A/dm² had the lowest value, and the plating (FIG. 2d)obtained at a current density of 1.5 A/dm² had the second lowest value.In contrast, the plating (FIG. 2b) obtained at a current density of 1.0A/dm² had a significant level of noise, and it was difficult to measurechanges in potential of neurons with high precision.

Example 3

[0077] Frequency characteristics of planar micro-electrode surfacescoated with electrolytic gold plating obtained at various currentdensities were compared with those of a conventional product. Electrodeshaving porous gold-plated surfaces obtained at a current density of 2.0A/dm² and 1.5 A/dm², respectively, had frequency characteristics similarto that of a conventional electrode having platinum black platingobtained by electrolysis. However, the gold plating obtained at acurrent density of 1.0 A/dm² had frequency characteristics significantlyinferior to those of the conventional product.

Example 4

[0078] A micro-electrode surface area was measured or calculated by thefollowing methods.

[0079] 1. Measurement by Gas Adsorption Method

[0080] The surface area of the micro-electrode having a porousgold-plated surface obtained in Example 1 at a current density of 1.5A/dm² was measured with a gas adsorption method using CO gas. As aspecimen to be measured, 64 gold-plated micro-electrodes provided on a1.3 mm×1.3 mm×1.1 mm glass substrate (hereinafter referred to as agold-plated micro-electrode block) were used. A single micro-electrodeis too small to be used as a specimen to be measured with the gasadsorption method. In addition, 64 platinum black-platedmicro-electrodes (hereinafter referred to as a platinum black-platedmicro-electrode block) provided on a glass substrate as described abovebut having platinum black plating by a conventional method (electrolyticplating) instead of gold plating, were used as comparative specimens.Note that each of the specimen blocks had a weight of 0.004 g. Theresults of the measurement are shown in Table 2. TABLE 2 Results ofMeasurement of Surface Area of Electrode having Gold-plated SurfaceSpecimen Surface area Gold-plated micro-electrode 0.02 m²/g or lessblock Platinum black-plated 20.2 m²/g micro-electrode block (comparativeexample)

[0081] The entire surface area of the gold-plated micro-electrode blockor the platinum black-plated micro-electrode block is represented by:

S′=(S−s)+αs

[0082] where S′ represents the entire surface area of the gold-platedmicro-electrode block or the platinum black-plated micro-electrode block(hereinafter referred to as a micro-electrode block); S represents theentire surface area of the micro-electrode block before forming platingon a 1.3 mm×1.3 mm×1.1 mm glass substrate; s represents the surface areaof a micro-electrode before plating (projection area), S′ represents theentire surface area of the gold-plated micro-electrode block or theplatinum black-plated micro-electrode block; as represents an increasein the surface area s of a micro-electrode by plating by a factor of α;and S−s, i.e., the surface area of the micro-electrode block excludingthe micro-electrode is not changed and only the surface area of themicro-electrode is increased by plating. The value of a is calculatedby:

α=(S′−S)/s+1.

[0083] In this case, the value of S is the surface area of a 1.3 mm×1.3mm×1.1 mm rectangular parallelepiped, i.e., 7.41 mm². The value of s(projection area) is the surface area of the 64 electrodes each having asize of 50 m×50 μm, i.e., 0.16 mm². Therefore, according to themeasurement result of S′ shown in Table 2, the value of α of theplatinum black-plated micro-electrode is calculated, to be 504955. Notethat the value of α (an increase in the surface area) of the gold-platedelectrode is below the detection limit of the gas adsorption method, andis estimated to be less than 455.

[0084] 2. Calculation of Surface Area of Micro-electrode

[0085] 2. 1. Impedance characteristics of Gold-plated Electrode

[0086] The impedance of the micro-electrode of Example 1 having a porousgold-plated surface obtained at a current density of 1.5 A/dm² wasmeasured by continuously changing frequency from 1 Hz to 100 kHz. Inthis measurement, a 0.3 mmφ platinum line was used as a counterelectrode and the measurement was conducted in 1.4 wt % NaCl aqueoussolution. The bias voltage was zero volts, and the amplitude of ameasuring voltage was 50 mV. The results are shown in FIG. 3. FIG. 3 isa Bode diagram, well-known to those skilled in the art, showing theresults of the impedance measurement, where the logarithm of theabsolute value of a measured impedance Z (i.e., log|Z|) and the phaseangle (θ) are plotted with respect to the logarithm of frequency f.Based on the Bode diagram, the measurement system is represented by an,equivalent circuit, thereby quantifying the surface area of themicro-electrode.

[0087] The measurement system including the micro-electrode can beestimated to be equivalent to a circuit actually including a capacitancegenerated between the ITO circuit pattern portion and a solution, aresistance generated on a micro-electrode interface, and the like, inaddition to the resistance of the solution, the resistance of the ITOcircuit pattern, a capacitance of an electric double layer in themicro-electrode surface, and the like, which are complicatedly connectedin series and parallel. For example, FIG. 4a shows an equivalent circuitof the measurement system including micro-electrode. The syntheticimpedance Z of the entire equivalent circuit shown in FIG. 4a can berepresented by: $\begin{matrix}{Z = {R_{1} + \frac{\begin{matrix}{{\left( {R_{2} + R_{3}} \right)\left( {1 - {\omega^{2}R_{2}R_{3}C_{1}C_{2}}} \right)} +} \\{\omega^{2}R_{2}R_{3}C_{1}\left\{ {{R_{3}C_{1}} + {\left( {R_{2} + R_{3}} \right)C_{2}}} \right\}}\end{matrix}}{\begin{matrix}{\left( {1 - {\omega^{2}R_{2}R_{3}C_{1}C_{2}}} \right)^{2} +} \\\left\lbrack {\omega \left\{ {{R_{3}C_{1}} + {\left( {R_{2} + R_{3}} \right)C_{2}}} \right\}} \right\rbrack^{2}\end{matrix}} - {j\quad \omega \frac{\begin{matrix}{{\left( {R_{2} + R_{3}} \right)\left\{ {{R_{3}C_{1}} + {\left( {R_{2} + R_{3}} \right)C_{2}}} \right\}} -} \\{R_{2}R_{3}{C_{1}\left( {1 - {\omega^{2}R_{2}R_{3}C_{1}C_{2}}} \right)}}\end{matrix}}{\begin{matrix}{\left( {1 - {\omega^{2}R_{2}R_{3}C_{1}C_{2}}} \right)^{2} +} \\\left\lbrack {\omega \left\{ {{R_{3}C_{1}} + {\left( {R_{2} + R_{3}} \right)C_{2}}} \right\}} \right\rbrack^{2}\end{matrix}}}}} & \lbrack I\rbrack\end{matrix}$

[0088] where R₁ represents the resistance of the ITO circuit patternportion (which does not contact the solution); R₂ represents theresistance of the ITO circuit pattern portion and the resistance of thesolution; R₃ represents the resistance of the micro-electrode surface;C₁ represents the capacitance of the electric double layer of themicro-electrode surface; C₂ represents the capacitance generated betweenthe ITO circuit pattern portion and the solution via an insulating film;and ω=2πf (ω: angular frequency and f: frequency). The absolute value|Z| of the above-described synthetic impedance and the phase θ arerepresented by |Z| (Z_(Re) ²+Z_(Im) ²)^(1/2) and θ=tan⁻¹(−Z_(Im)/Z_(Re))(where Z_(Re) is the real part of Z and Z_(Im) is the imaginary part ofZ), respectively.

[0089] Here, the equivalent circuit shown in FIG. 4a was simulated bycalculating the synthetic impedance of the equivalent circuit bychanging R₁, R₂, C₁ and C₂ to select a combination of R₁, R₂, R₃, C₁ andC₂ which provide a Bode diagram most approximate to the Bode diagramshown in FIG. 3. FIG. 5 is a Bode diagram where R₁=200 Ω, R₂=5 kΩ, R₃=5MΩ, C₁=0.01 μF and C₂=100 pF. It is apparent that the results shown inFIG. 5 are in agreement with the results of the actual measurement shownin FIG. 3 to an excellent extent.

[0090] Here, in the measurement system including the micro-electrode,R₃>>R₁, R₃>>R₂ and C₂<<C₁. Therefore, if it is assumed that R₃→∞ andC₂→0, the above-described expression [I] can be approximated by:$\begin{matrix}{{\lim\limits_{R_{3}->\infty}{\lim\limits_{C_{2}->0}Z}} = {{R_{1} + R_{2} - {j\frac{1}{\omega \quad C_{1}}}} = {R_{1} + R_{2} + \frac{1}{j\quad \omega \quad C_{1}}}}} & \lbrack{II}\rbrack\end{matrix}$

[0091] If R₁+R₂=R and C₁=C, the measurement system including themicro-electrode can be approximated by a simple circuit shown in FIG.4b. Hereinafter, analysis was further conducted using the equivalentcircuit shown in FIG. 4b.

[0092] The synthetic impedance Z of the entire equivalent circuit shownin FIG. 4b is represented by Z=R+(jωC)⁻¹ where R represents theresistance of a circuit pattern; C represents the resistance of theelectric double layer of the micro-electrode surface; and ω=2πf (ωrepresents angular frequency and f represents frequency). The absolutevalue |Z| of the above-described synthetic impedance and the phase θ arerepresented by |Z|=(R²+(1/ωC)²)^(1/2) and θ=tan⁻¹(−ωC/R), respectively.

[0093] The equivalent circuit shown in FIG. 4b was simulated bycalculating the synthetic impedance of the equivalent circuit bychanging the values of R and C to select a combination of R and C whichprovides a Bode diagram which is most approximate to the Bode diagramshown in FIG. 3. FIG. 6 is a Bode diagram where R=5 kΩ and C=0.01 μF. Itis apparent that the results shown in FIG. 6 are in agreement with theresults of the actual measurement shown in FIG. 3 to an excellentextent.

[0094] Next, FIG. 7 is a graph showing the actually measured values ofthe impedance of a micro-electrode used as a control, which does nothave a gold-plated surface. The measurement conditions are the same asused in measuring the impedance of the multiple electrode having theabove-described gold-plated surface (FIG. 3).

[0095] Next, FIG. 8 shows the results of the synthetic impedance of theequivalent circuit obtained by simulation of the equivalent circuitshown in FIG. 4b where R=5 kΩ and C=250 pF. It is apparent that theresults shown in FIG. 8 are in agreement with the results of the actualmeasurement shown in FIG. 7 to an excellent extent.

[0096] Here, it is assumed that the capacitance of the electric doublelayer of a micro-electrode having a gold-plated surface is representedby C_(A) and the capacitance of the electric double layer of themicro-electrode before gold plating treatment is represented by C_(B).According to the above-described simulation results, the electrostaticcapacities C_(A) and C_(B) of the micro-electrode are 0.01 μF and 250pF, respectively, whereby a relationship C_(A)=40C_(B) is obtained.Here, generally, electrostatic capacity C_(ap) is represented byC_(ap)=ε₀ε_(r)S/d (ε₀: the dielectric constant of a vacuum; ε_(r): therelative dielectric constant of a dielectric material; S: the surfacearea of an electrode; and d : the thickness of the dielectric material).The value of electrostatic capacity C_(ap) is proportional to thesurface area of the electrode. Therefore, the above-describedrelationship indicates that the surface area of a micro-electrode havinga gold-plated surface is increased by a factor of 40 by the provision ofthe gold plating.

[0097]FIG. 9 is a graph showing the actually measured values of theimpedance of a micro-electrode having a platinum black-plated surface ina manner similar to a micro-electrode having a gold-plated surface. Themeasurement conditions are the same as used in measuring the impedanceof a micro-electrode having a gold-plated surface (FIG. 3). Next, FIG.10 shows the result of the synthetic impedance of the equivalent circuitshown in FIG. 4b obtained by simulation where R=5 kΩ and C=0.05 μF. Itis apparent that the results shown in FIG. 10 are in agreement with theresults of the actual measurement shown in FIG. 9 to an excellentextent.

[0098] Similarly, it is assumed here that the capacitance of theelectric double layer of a micro-electrode having a gold-plated surfaceis represented by C_(A) and the capacitance of the electric double layerof the micro-electrode before gold plating treatment is represented byC_(B). According to the above-described simulation results, theelectrostatic capacities C_(A) and C_(B) of the micro-electrodes are0.01 μF and 0.05 μF, respectively, whereby a relationship C_(A)=200C_(B)is obtained. This relationship indicates that the surface area of amicro-electrode having a gold-plated surface is increased by a factor of200 by the provision of the gold plating.

[0099] According to this simulation, in order to obtain an impedance of50 kΩ or less, which is the limit of the impedance of a micro-electrode,the surface area needs to be increased by a factor of 10 or more usinggold plating, compared to the projection area.

Example 5

[0100] A planar multiple electrode having porous gold plating obtainedby electrolysis at a current density of 1.5 A/dm² was subjected to alifetime test in comparison with a conventional electrode (platinumblack plating). In the lifetime test, an experiment similar to a typicalacute experiment was repeated. Specifically, an experiment as describedin Example 2 was repeated. After the end of each experiment, theelectrode was treated with collagenase (20 u/ml) overnight and a cellslice specimen was peeled off, followed by washing with distilled water.Subsequently, the impedance of the micro-electrode was measured. FIG. 11is a graph in which the impedance changes after the end of eachexperiment are plotted. According to the result, an increase andvariation in the impedance with an increase in the number of times ofuse is smaller than that of conventional plating (platinum blackplating).

[0101] In contrast, the impedance of a conventional product having aplatinum black-plated surface by electrolysis was significantlyincreased after 17 to 18 cycles as shown in FIG. 11. As described above,an electrode having porous gold plating has a higher level ofrecyclability, and the potential change of the electrode can be stablymeasured, as compared to conventional products.

[0102] Although the present invention is described with reference to theabove-described examples, the present invention is not limited to theseexamples. The present invention may be implemented to modified,improved, and changed embodiments based on the knowledge of thoseskilled in the art without departing the scope of the present invention.

INDUSTRIAL APPLICABILITY

[0103] According to the present invention, a multiple electrode forextracellular recording is provided which has a high strength, iscapable of recording the electrical activities of neurons for a longperiod of time, and is easy to recycle. A plurality of micro-electrodesincluded in the multiple electrode have a low level of impedance withoutloss of the strength by utilizing porous gold plating. Therefore, it iseasy to apply a constant current stimulus to the multiple electrode, andthe multiple electrode is preferably suited to monitor a response of acultured cell to an electrical stimulus.

1. A multiple electrode for measuring electro-physiologicalcharacteristics of a cell, the electrode comprising: a plurality ofmicro-electrodes provided on a substrate; and a wiring portion forproviding an electrical signal to the micro-electrodes or extracting anelectrical signal from the micro-electrodes, wherein themicro-electrodes have a porous conductive material on a surface thereof,the conductive material is selected from the group consisting of gold,titanium nitride, silver oxide, and tungsten, and the impedance of eachof the micro-electrode is 50 kΩ or less.
 2. A multiple electrodeaccording to claim 1, wherein the porous conductive material is gold,and is provided by passage of current at a current density of 1.0 to 5.0A/dm² for 10 to 360 sec.
 3. A multiple electrode for measuringelectro-physiological characteristics of a cell, the electrodecomprising: a plurality of micro-electrodes provided on a substrate; anda wiring portion for providing an electrical signal to themicro-electrodes or extracting an electrical signal from themicro-electrodes, wherein the surface area of the micro-electrodecalculated from an electrostatic capacity of an equivalent circuithaving substantially the same impedance as that of the micro-electrode,is greater than or equal to at least 10 times and less than 200 timesthe projection area of the micro-electrode, and the impedance of each ofthe micro-electrode is 50 kΩ or less.
 4. A multiple electrode accordingto claim 3, wherein a surface area of the micro-electrode measured by agas adsorption method is less than or equal to 5×10⁵ times theprojection area of the micro-electrode.
 5. A multiple electrodeaccording to any of claims 1 to 4, wherein the micro-electrodes arearranged on the substrate in a form of a matrix, the wiring portionincludes a lead line connected to the micro-electrode and an electricaljunction connected to an end of the lead line, and at least a surface ofthe lead line is covered with an insulating layer.
 6. A multipleelectrode according to any of claims 1, 3, 4 and 5, wherein the porousconductive material is provided by etching.
 7. An integrated cellinstaller comprising a multiple electrode according to any of claims 1to 6, wherein the integrated cell installer has a cell installing regionfor placing a cell or tissue on the substrate of the multiple electrode.8. A cellular potential measuring apparatus comprising: an integratedcell installer according to claim 7; an output signal processorconnected to the micro-electrodes for processing an output signal due toan electro-physiological acitivity of a cell or tissue; and a stimulussignal provider for optionally providing an electrical stimulus to thecell or tissue.
 9. A cellular potential measuring system comprising: acellular potential measuring apparatus according to claim 8; an opticalmonitoring apparatus for optically monitoring a cell or tissue; and/or acell culture apparatus for controlling the culture environment of thecell or tissue.